The present invention relates to the introduction of DNA and RNA sequences into a mammalian cell to achieve controlled expression of a polypeptide. It is therefore useful in gene therapy, vaccination, and any therapeutic situation in which a polypeptide should be administered to a host or cells of said host, as well as for the production of polypeptides by mammalian cells, e.g., in culture or in transgenic animals.
Gene therapy is a set of approaches for the treatment of human disease based on the transfer of genetic material (DNA/RNA) into an individual. Gene delivery can be achieved either by direct administration of gene-containing viruses or DNA to blood or tissues, or indirectly through the introduction of cells manipulated in the laboratory to harbor foreign DNA (see, e.g. U.S. Pat. No. 5,399,346). The tremendous promise of gene therapy is stymied by inefficient gene transfer. G. Parmiani, F. Arienti, J. Sule-Suso, C. Melani, M. P. Colombo, V. Ramakrishna, F. Belli, L. Mascheroni, L. Rivoltini and N. Cascinelli, Cytokine-based gene therapy of human tumors, An Overview, Division of Experimental Oncology D, Instituto Nazionale Tumori, Milan, Italy, Folia Biol (Phraha) 42: 305-9 (1996); P. Hess, Gene therapy: a review, American Association of Clinical Chemistry, Laboratory Corporation of America, Louisville, Ky., 40213, USA, Clin Lab Med 16: 197-211 (1996); and N. Miller and R. Vile, Targeted vectors for gene therapy, Laboratory of Cancer Gene Therapy, Rayne Institute, St. Thomas' Hospital, London, United Kingdom, FASEB J 9: 190-9 (1995).
The clinical application of gene therapy, as well as the utilization of recombinant retrovirus vectors, has been delayed because of safety considerations. Integration of exogenous DNA into the genome of a cell can cause DNA damage and possible genetic changes in the recipient cell that could predispose to malignancy. A method which avoids these potential problems would be of significant benefit in making gene therapy safe and effective.
Vaccination with immunogenic proteins has eliminated or reduced the incidence of many diseases; however there are major difficulties in using proteins associated with other pathogens and disease states as the immunogen. Many protein antigens are not intrinsically immunogenic. More often, they are not effective as vaccines because of the manner in which the immune system operates.
The immune system of mammalians consists of several interacting components. The best characterized and most important parts are the humoral and cellular branches. Humoral immunity involves antibodies, proteins which are secreted into the body and which directly recognize an antigen. The cellular system, in contrast, relies on special cells which recognize and kill other cells which are producing foreign antigens. This basic functional division reflects two different strategies of immune defense. Humoral immunity is mainly directed at antigens which are exogenous to the animal whereas the cellular system responds mainly to antigens which are actively synthesized within the cells of the animal.
Antibody molecules, the effectors of humoral immunity, are secreted by special cells, B cells, in response to antigen. Antibodies can bind to and inactivate antigen directly (neutralizing antibodies) or activate other cells of the immune system to destroy the antigen.
Cellular immune recognition is mediated by a special class of lymphoid cells, the cytotoxic T cells. These cells do not recognize whole antigens but instead they respond to degraded peptide fragments thereof which appear on the surface of the target cell bound to proteins called class I major histocompatibility complex (MHC) molecules. Essentially all nucleated cells have class I molecules. It is believed that proteins produced within the cell are continually degraded to peptides as part of normal cellular metabolism. These fragments are bound to the MHC molecules and are transported to the cell surface. Thus the cellular immune system is constantly monitoring the spectra of proteins produced in all cells in the body and is poised to eliminate any cells producing foreign antigens.
A large number of disease states can benefit from the administration of therapeutic and/or prophylactic polypeptides. Such polypeptides include e.g. lymphokines, such as interleukins, tumor necrosis factor, the interferons; growth factors, such as nerve growth factor, and human growth hormone; tissue plasminogen activator; factor VIII:C; granulocyte-macrophage colony-stimulating factor; erythropoietin; insulin; calcitonin; thymidine kinase; and the like. Moreover selective delivery of toxic peptides (such as ricin, diphtheria toxin, or cobra venom factor) to diseased or neoplastic cells can have major therapeutic benefits.
Vaccination by intramuscular injection of antigen-encoding DNA is a promising approach (J. J. Donnelly, J. B. Ulmer, M. A. Liu, J. Immunol. Methods 176, 145 (1994); R. M. Conry et al., Cancer Res. 54, 1164 (1994); C. H. Hsu et al., Nature Med. 2, 540 (1996); R. E. Tascon et al., Nature Med. 2, 888 (1996)), but how an immune response is accomplished is not fully understood, although bone marrow-derived antigen presenting cells (APC), rather than myocytes, seem to induce the immune responses after migration to the spleen (M. Corr et al., J. Exp. Med. 184, 1555 (1996)). Intramuscular injection of pure plasmid DNA into the host still poses several problems: (i) The efficiency of the DNA-uptake seems to be quite low and dose-dependent, which means that a large amount of plasmid DNA has to be injected to elicit a protective immune response (R. R. Deck et al., Vaccine, 15, 71 (1997)). This in turn might lead to adverse effects through immune stimulation by bacterial DNA-sequences (D. S. Pisetsky, J. Immunol. 156, 421 (1996)). (ii) Intramuscular DNA injection does not seem to induce immune responses at distant mucosal surfaces (R. R. Deck et al., Vaccine, 15, 71 (1997)). (iii) There are only low numbers of antigen-presenting cells (APC) in the muscle tissue and thus protection against infectious agents after intramuscular injection of plasmid DNA may only be possible with immunologically very potent antigens. This makes it desirable to deliver the antigen-encoding DNA directly to splenic APC.
Recently, attenuated Shigella flexneri (D. R. Sizemore, A. A. Branstrom, J. C. Sadoff, Science 270, 299 (1995)) and invasive Escherichia coli (P. Courvalin, S. Goussard, C. Grillot-Courvalin, Life Sciences 318, 1207 (1995)) were used for plasmid delivery in cultured mammalian cells, in guinea pigs and in mice. Shigella flexneri and E. coli are Gram-negativ bacteria, though, which contain Lipopolysaccharide (LPS), exhibiting strong endotoxic effects in mammals. Furthermore, these bacteria are only suiteable for the introduction of therapeutic molecules into certain cell types, e.g., enterocytes (P. J. Sansonetti. Pathogenesis of shigellosis. Curr. Top. Microbiol. Immunol., 180:1-143(1992).
Thus, new techniques were needed to solve the above-described problems associated with immunization, gene therapy, and delivery of therapeutic polypeptides to cells, both ex vivo and in vivo.